Human intestinal absorption (HIA) of ionizable compounds can depend simultaneously on three key properties: solubility, permeability, and pKa (Avdeef A., “Absorption and Drug Development”, Wiley Interscience, NY, 2003). This association is exemplified by the Absorption Potential (Dressman J B et al., “J. Pharm. Sci.”, 1985, 74, 588), the Biopharmaceutics Classification System (Guidance for Industry, “Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System”, FDA, Washington, D.C., USA, August 2000), and the Maximum Absorbable Dose function (Curatolo W. “Pharm. Sci. Tech. Today”, 1998, 1, 387). In the simplest terms, Fick's laws of diffusion underlie all of these models.
In the intestine, water-soluble weak bases are better absorbed from slightly alkaline regions (e.g., in the distal ileum), and weak acids are better absorbed from slightly acidic regions (e.g., proximal jejunum). This was rationalized by Brodie and coworkers (Shore P A et al., “J. Pcol. Exp. Therap.”, 1957, 119, 361), who introduced the pH Partition Hypothesis to explain the influence of pH on the intestinal absorption of ionizable drugs. Rat intestines were perfused in situ with a drug solution of varied pH. At the same time, the drug was injected intravenously. The concentration of the drug in the luminal perfusate was adjusted until there was no net transport across the intestinal wall, so that it was possible to define the blood-lumen barrier ratio
                    D        =                                            [              drug              ]                        BLOOD                                              [              drug              ]                        LUMEN                                              (        1        )            
If only the neutral form of the drug permeates, then equation (1) can be predicted from the pKa of the drug and the pH gradient between the two sides of the intestinal barrier (Shore P A et al., “J. Pcol. Exp. Therap.” 1957, 119, 361):
                    D        =                              (                          1              +                              10                                                      -                                          pK                      a                                                        +                                      p                    ⁢                                                                                  ⁢                                          H                      BLOOD                                                                                            )                                (                          1              +                              10                                                      -                                          pK                      a                                                        +                                      p                    ⁢                                                                                  ⁢                                          H                      LUMEN                                                                                            )                                              (        2        )            
Equation (2) is derived from the pH dependence of permeability, based on the well known Henderson-Hasselbalch (HH) equation. Direct measurement of in situ intestinal perfusion absorption rates confirmed the pH dependence, further supporting that theory and observation were well matched in these early experiments.
The pH Partition Hypothesis suggests that membrane permeability will be highest at the pH where the molecule is least charged. But this is also the pH where the molecule is least soluble. It is particularly important to note that in Brodie's work, all of the compounds tested have relatively high water solubility. At the site of absorption, the amount of the uncharged species and the tendency of the neutral species to cross the phospholipid membrane barrier are both important predictors of absorption. The intrinsic permeability coefficient, Po, characterizes the membrane transport of the uncharged species. The concentration of the uncharged species, Co, depends on the dose, the solubility, the pKa of a molecule, and the pH at the site of absorption, according to the HH equation.
Combinatorial chemistry programs have tended to select for higher molecular weight molecules, which are predictably low in solubility. ‘Early warning’ tools, such as Lipinski's ‘Rule of 5’ (Lipinski C., “Amer. Pharm. Rev.” 2002, 5, 82), and computer programs that predict solubility from 2-D structure, attempt to weed out such molecules early in discovery programs. Still, many solubility-problematic molecules remain unrecognized, due to the overly simplistic early methods used to measure solubility, and the masking effect of organic solvents (e.g., dimethyl sulfoxide) used in discovery measurements. Arguably, nephelometry-based kinetic solubility measurements, although fast, are no more reliable than in silico prediction methods (Glomme A. et al. “J. Pharm. Sci.”, 2005, 94, 1).
Measurement of solubility of sparingly soluble molecules, e.g. compounds or drugs, is challenging for a number of reasons. Notably, the HH equation only poorly predicts the pH dependence of sparingly soluble molecules (Bergström CAS et al., “Eur. J. Pharm. Sci.”, 2004, 22, 387 and U.S. Pat. No. 6,569,686 B2), largely due to the prevalence of aggregates and micelle-like structures in solution. Such aggregates have unusually high solubility (in pH solutions where charged species persist), with a sensitive temperature dependence.
Permeability measurement is also fraught with substantial uncertainty, since results depend particularly on how assay pH, the aqueous boundary layer (ABL), and incomplete mass balance are treated in such assays (both cellular and artificial membrane permeability assay) by different laboratories (Avdeef A. et al., “Eur. J. Pharm. Sci.”, 2005, 24, 333).
Thus, more accurate (but still fast) solubility, and permeability methods in the candidate selection stage in pharmaceutical research and development would be particularly helpful in recognizing at a much earlier time truly problematic molecules (Bergström CAS et al., “Eur. J. Pharm. Sci.”, 2004, 22, 387 and Glomme A. et al. “J. Pharm. Sci.”, 2005, 94, 1).
Besides the described effects of pH, solubility, and permeability on absorption processes, particularly on HIA processes, the use of excipients can essentially affect absorption processes, particularly absorption processes of sparingly soluble molecules. Taking into consideration the complexity of the above mentioned effects, the evaluation of suitable excipients being capable of optimizing the absorption processes is a very difficult task. Today, such evaluation is performed by conducting animal experiments. Animal experiments usually are comparably time consuming, cause comparably large efforts and are ethically controversial.
Therefore, there is a need for an ethically passable method allowing a fast, compound sparing, cost effective, and reasonably accurate prediction of absorption properties of sparingly soluble molecules, i.e. low solubility compounds or drugs, taking into account the effect of excipients on said absorption.